ProjectSummary The NF- ?B dimers bind to specific DNA response elements known as the ?B DNA sites located in the promotersandenhancersofthousandsofgenes,andregulatetheirexpression.Althoughtheroughly10bp long ?B sequences follow a consensus,hundreds of specific sequences can fit the consensus. Sequence variations can result in differences in NF-?B-DNA binding affinity, kinetics and conformations leading to changes in transcriptional output. Indeed, other and we reported that as few as a single bp change can havesevereeffectingeneregulationbytheNF-?Bdimers.AffinitiesoftheNF-?B:DNAcomplexesderived from in vitro experiments do not always correlate with in vivo binding and gene regulation. These observations suggest that there are modulators present in the cell and without their inclusion in in vitro experiments in vivo and in vitro results will not reconcile. On the other hand, without proper in vitro experimental set up, proper investigation of regulatory mechanisms in vivo is difficult to accomplish. Cellular experiments performed over the past 10 years established the presence of several of such modulators, but their mechanisms of action could not be properly explained without thorough biochemical and biophysical experiments. We term these modulators cofactors. These cofactors alter the DNA binding affinity of NF-?B p50:RelA heterodimer and RelA homodimers in a ?B sequence-specific manner. The focus of this proposal is to use new experiments to propose a unifying principle of how the cofactors regulateNF-?Bactivity. We propose that when the affinity between an NF-?B:?B DNA complex is weak, a cofactor can act positively enhancing the affinity of NF-?B:DNA complexes by directly contacting NF-?B without contacting DNA allowing geneexpression tooccur.Alternatively,a cofactor canact negatively by removing NF-?B off the DNA (or reduce affinity). Several positive cofactors and few negative cofactors are known. We will investigate the mode of actions of a few of these cofactors in vitro. Specifically, we will identify the site of interactionofboth positive and negative cofactors on RelAandhowtheyuse allosteric mechanismtoalter DNAbindingbyRelA.Sincenocofactorspecifictop50isknown,wealsoplantoidentifycofactorsspecific tothep50subunitandinvestigateifandhowthesenewcofactorsacttogetherwithRelA-specificcofactors. We will generate mutants of RelA defective in cofactor binding and test how gene expression profile and DNAbindingincellsaltersinresponsetospecificstimulus.